Toxicological assessment of benzalkonium chloride using planaria mobility: A comparison of manual and digital tracking methods.

Abstract

The principle of the 3Rs-Reduction, Refinement, and Replacement-encourages minimizing animal use, improving experimental design, and developing alternative models for toxicology testing. Among such models, planaria (aquatic flatworms) have gained increasing attention in pharmacology, regenerative medicine, and toxicology because of their simple anatomy, high environmental sensitivity, exceptional regenerative ability, and ease of laboratory maintenance. In this study, we examined the effects of benzalkonium chloride (BAC)-a commonly used pharmaceutical excipient with antimicrobial and permeability-enhancing properties, as well as a known environmental toxicant-on the locomotor activity of Schmidtea mediterranea using both manual assessment and Lolitrack video-tracking software. Six concentrations of BAC (5-1000 μg/mL) and a negative control were tested. Both approaches showed an overall reduction in locomotor activity over time, though manual analysis indicated a transient stimulation at lower concentrations. The software-based method demonstrated greater reliability, precision, and objectivity, making it preferable for toxicity evaluation in planaria.